Adoptive immunotherapy, which involves the transfer of autologous antigen-specific T cells generated ex vivo, is a promising strategy to treat viral infections and cancer. The T cells used for adoptive immunotherapy can be generated either by expansion of antigen-specific T cells or redirection of T cells through genetic engineering (Park, Rosenberg et al. 2011). Transfer of viral antigen specific T cells is a well-established procedure used for the treatment of transplant associated viral infections and rare viral-related malignancies. Similarly, isolation and transfer of tumor specific T cells has been shown to be successful in treating melanoma.
Novel specificities in T cells have been successfully generated through the genetic transfer of transgenic T cell receptors or chimeric antigen receptors (CARs) (Jena, Dotti et al. 2010). A schematic representation for the 3 generations of CARs is presented in FIG. 1. CARs are synthetic receptors consisting of a targeting moiety that is associated with one or more signaling domains in a single fusion molecule. In general, the binding moiety of a CAR consists of an antigen-binding domain of a single-chain antibody (scFv), comprising the light and variable fragments of a monoclonal antibody joined by a flexible linker. Binding moieties based on receptor or ligand domains have also been used successfully. The signaling domains for first generation CARs are derived from the cytoplasmic region of the CD3zeta or the Fc receptor gamma chains. First generation CARs have been shown to successfully redirect T-cell cytotoxicity. However, they failed to provide prolonged expansion and anti-tumor activity in vivo. Signaling domains from co-stimulatory molecules, as well as transmembrane and hinge domains have been added to form CARs of second and third generations, leading to some successful therapeutic trials in humans, where T-cells could be redirected against malignant cells expressing CD19 (June et al., 2011). However, the particular combination of signaling domains, transmembrane and co-stimulatory domains used with respect to CD19 ScFv, was rather antigen-specific and cannot be expanded to any antigen markers.
Chronic lymphocytic leukemia (CLL) is one of the most commonly diagnosed leukemias managed by practicing hematologists. For many years patients with CLL have been viewed as similar, with a long natural history and only marginally effective therapies that rarely yielded complete responses. Recently, several important observations related to the biologic significance of VH mutational status and associated ZAP-70 overexpression, disrupted p53 function, and chromosomal aberrations have led to the ability to identify patients at high risk for early disease progression and inferior survival. Concurrent with these investigations, several treatments including the nucleoside analogues, monoclonal antibodies rituximab and alemtuzumab have been introduced. Combination of these therapies in clinical trials has led to high complete and overall response rates when applied as initial therapy for symptomatic CLL. Thus, the complexity of initial risk stratification of CLL and treatment has increased significantly. Furthermore, when these initial therapies do not work, approach of the CLL patient with fludarabine-refractory disease can be quite challenging (Byrd J. C et al, 2014).
One candidate antigen of immunotherapies for chronic lymphocytic leukemia (CLL) is Tyrosine-protein kinase transmembrane receptor ROR1 (also called NTRKR1; UniProtKB/TrEMBL) entries: Q01973). ROR1 (The receptor tyrosine kinase-like orphan receptor 1) is a 120-kDa glycoprotein containing an extracellular immunoglobulin (Ig)-like, Kringle, and Frizzled-like cysteine rich domain (FIG. 2). The protein encoded by this gene is a receptor tyrosine kinase that modulates neurite growth in the central nervous system. It is a type I membrane protein and belongs to the ROR subfamily of cell surface receptors (Reddy et al, 1997). Although ROR1 protein expression in patients with CLL with respect to normal leukocytes and its role in the pathobiology of CLL merits further studies. ROR1 may be an appropriate target for cancer immunotherapy (Daneshmanesh et al; 2008). ROR1 is indeed expressed on a variety of B-cell malignancies, but also on subsets of some solid tumors, including breast, colon, lung, and kidney tumors. It is believed that ROR1 functions in oncogenic signaling to promote tumor cell survival in epithelial tumors. Importantly, ROR1 is not expressed on vital organs, except adipose and pancreatic tissue, which reduces potential toxicities from killing of normal cells (Hudecek et al, 2013). ROR1 is expressed during embryogenesis but absent from normal adult tissues, apart from a subset of immature B-cell precursors, and low-level expression on adipocytes (Hudecek et al., 2010; Matsuda et al., 2001). ROR1 was first shown to be expressed in B-cell chronic lymphocytic leukemia (B-CLL) by transcriptional profiling (Klein et al., 2001; Rosenwald et al., 2001) and was subsequently identified on the surface of many cancers including mantle cell lymphoma (MCL), acute lymphoblastic leukemia (ALL) with a t(1;19) chromosome translocation, and a subset of lung, breast, colon, pancreas, renal, and ovarian cancers (Baskar et al., 2008; Bicocca et al., 2012; Daneshmanesh et al., 2008; Dave et al., 2012; Fukuda et al., 2008; Yamaguchi et al., 2012; Zhang et al., 2012a, 2012b). In both lung adenocarcinoma and t(1;19) ALL, ROR1 cooperates in oncogenic signaling and knockdown of ROR1 with siRNA exposed a critical role for this molecule in maintaining tumor cell survival (Bicocca et al., 2012; Choudhury et al., 2010; Gentile et al., 2011; Yamaguchi et al., 2012). Thus, ROR1 loss may not be readily tolerated by tumors making it an attractive candidate for CAR directed T-cell therapy that could be broadly applied. The present inventors have thus considered that ROR1 could be a valuable target antigen for treating CLL as well as solid tumors such as breast, colon, lung, ovarian and kidney tumors, by using CAR-expressing T cells.
The laboratories of Dr. Stanley Riddell and Dr. Laurence Cooper have previously engineered and validated anti-ROR1 scCARs containing the 4A5 and the 2A2 scFvs, respectively (Cooper et al 2010; Hudecek et al., 2013). In particular, Hudecek et al discloses anti-ROR1 scCARs which contain an IgG4 hinge of diverse length and a CD28 transmembrane domain.
There is still the need for the improvement of CAR functionality by designing CAR architecture and using suitable components since these parameters play a role important and a fine tuning is necessary.
Therefore, as an alternative to the previous strategies, the present invention provides with ROR1 specific CARs, which can be expressed in immune cells to target ROR1 malignant cells with significant clinical advantage. The inventors have found that, by combining CAR architecture to the choice of suitable components, they could obtain specific ROR1 single chain CARs with high cytotoxicity towards cancerous target cells,